Compositions and methods for fluid biopsy of melanoma

ABSTRACT

The present invention provides methods for identifying circulating melanoma cells (CMCs) in a biological sample and methods for diagnosing metastatic melanoma in a subject. The methods disclosed can be used on non-enriched blood samples to identify CMC using detectable agents that are specific for a biomarker of CMCs and assessing the morphology of the cells having the detectable agents. The presence or absence of a detectable agent in combination with morphological characteristics of the cells can be used diagnose a subject with metastatic melanoma based on the number of CMCs is present in the sample.

This application claims the benefit of priority of U.S. ProvisionalApplication No. 61/991,088, filed May 9, 2014, the entire contents ofwhich is incorporated herein by reference.

The present invention relates generally to the field of cancerdiagnostics, and more specifically to methods for diagnosing metastaticmelanoma.

BACKGROUND OF THE INVENTION

Circulating tumor cells (CTCs) released from either a primary tumor orits metastatic sites hold important information about the biology of thetumor. CTCs can be considered not only as surrogate biomarkers formetastatic disease but also as a promising key tool to track tumorchanges, treatment response, cancer recurrence or patient outcomenon-invasively. However, the extremely low levels of CTCs in thebloodstream combined with their unknown phenotype has significantlyimpeded their detection. Thus, the research to evaluate the clinicalutility of CTCs has been limited. A variety of technologies have beendeveloped over the past 20 years for specific isolation of CTCs in orderto utilize their information.

In the context of melanoma, one of the more widely used techniques fordetecting circulating melanoma cells (CMCs) is an RT-PCR method thatdetects the presence of tumor RNA in the bloodstream of melanomapatients. Recently, methodologies have been developed for detecting CMCsthat rely on enrichment of CMCs in a sample by capturing the intactCMCs, allowing downstream molecular, morphologic and/or phenotypiccharacterization. One frequently used method depends on immunomagneticenrichment of tumor cells from peripheral blood. However, one of themain limitations of this positive selection is that only CMCs withsufficient expression of the selected surface marker are detected. Onenegative selection approach relies on anti-CD45-coated immunomagneticbeads to selectively remove peripheral blood mononuclear cells (PBMCs).Still other enrichment-based methodologies rely on physical differencesbetween PBMCs and CMCs such as size or density. However, technicalinadequacies of these methodologies, which include low recovery rates,in combination with few suitable biomarkers that are expressed by allCMCs, has limited their adoption.

Thus, there exists a need for improved methods for CMC detection andcharacterization. The present disclosure addresses this need as well asproviding related advantages.

SUMMARY OF INVENTION

The present invention provides methods for identifying CMCs in abiological sample and methods for diagnosing metastatic melanoma in asubject.

The methods for identifying CMCs can include the steps of: (a)contacting a biological sample of non-enriched blood with one or moredetectable agents, wherein at least one of the one or more detectableagents is specific for a biomarker of CMCs; (b) determining the presenceor absence of the one or more detectable agents in or on nucleated cellsin the sample; and (c) assessing the morphology of the nucleated cellshaving the one or more detectable agents, wherein the CMCs areidentified based on a combination of the presence or absence of the oneor more detectable agents and morphological characteristics of thenucleated cells.

The methods for diagnosing metastatic melanoma can include the steps of:(a) contacting a biological sample of non-enriched blood with one ormore detectable agents, wherein the sample was obtained from a subjectsuspected of having metastatic melanoma or diagnosed with havingmelanoma, wherein at least one of the one or more detectable agents isspecific for a biomarker of CMCs; (b) determining the presence orabsence of the one or more detectable agents in or on nucleated cellspresent in the sample; (c) assessing the morphology of the nucleatedcells having the one or more detectable agents; and (d) identifying thepresence of CMCs in the sample based on a combination of the presence orabsence of the one or more detectable agents and morphologicalcharacteristics of the nucleated cells, wherein the subject is diagnosedwith metastatic melanoma when a predetermined number of CMCs is presentin the sample.

In additional embodiments, CMCs can be identified by automatedfluorescent microscopy. In some aspects, the methods compriseimmunofluorescent staining of nucleated cells with antibodies orfunctional fragments thereof that specifically bind biomarkers for CMCsand, in some aspects, surrounding white blood cells (WBCs) in thesample. In additional embodiments, the CMCs include distinctimmunofluorescent staining from surrounding nucleated cells. In furtherembodiments, the CMCs comprise distinct morphological characteristicscompared to surrounding nucleated cells.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show CSPG4 expression in melanoma cells. (FIG. 1A)Representative merged images of WM1617, WM278 and WM1789 melanoma cellsstained with CSPG4-specific mAbs are shown in column 1, 2 and 3 (fromleft to right), respectively. Controls are shown in line 1, thecombination of the 7 CSPG4-specific mAbs is shown in line 2 and the useof each CSPG4-specific mAb individually is shown from line 3 to 9. Lightgrey is CSPG4. (FIG. 1B) Relative CSPG4 intensity normalized against thebackground signal on PBMCs in WM1678, WM278 and WM1789 melanoma celllines.

FIGS. 2A-2C show the detection of melanoma cells spiked into PBMCs fromnormal blood. Melanoma cells from 3 melanoma cells lines (FIG.2A—WM1617, FIG. 2B—WM278 and FIG. 2C—WM1789) were spiked at differentconcentrations into PBMCs from a normal blood donor to produce 6 slidesin triplicate with 0, 10, 25, 50, 100 and 500 melanoma cells per slideand for each melanoma cell line. The expected number of cells spiked isplotted versus the number of cells detected.

FIG. 3 shows a scatter plot of candidate cells used to define CMCs. Tennormal blood donor, 40 melanoma patients and 3 melanoma cell linesclassified by relative CSPG4 protein expression and relative nuclearsize are shown. Both measurements are normalized against the valuesobtained in surrounding PBMCs. Cells from melanoma patients, normaldonors and cell lines are represented in black, light grey and grey,respectively.

FIGS. 4A-4E show the prevalence of CMCs in metastatic melanoma patients.(FIG. 4A) Metastatic melanoma patients (n=40) and normal blood donors(n=10) were evaluated using the HD-CTC platform in combination with apanel of 7 mAbs. The mean values are shown as a plain black line. P<0.05Wilcoxon t test. (FIG. 4B) HD-CMC/ml was calculated for each patient.Dark bars indicate the amount of CSPG4 bright HD-CMCs and light barsindicate the amount of dim CSPG4 HD-CMCs detected per melanoma patient.(FIG. 4C) Representative merged images of 8 HD-CMCs from 2 melanomapatients. Hoechst staining is represented in dark grey, CSPG4 in lightgrey and CD45 in bright grey. (FIG. 4D) Roundness (perimetersquared)/(4*pi*area), with 1 indicating perfect circle and larger valuesindicating oblong objects. (FIG. 4E) Area comparisons between PBMCs andHD-CMC from melanoma patients. The mean values are shown as a line.Error bars represent the standard deviation.

FIGS. 5A-5B show the characterization of CMCs with HMB45. (FIG. 5A)Scatter plot of HD-CMCs detected in 40 melanoma patients classified byrelative CSPG4 signal intensity and relative HMB-45 signal intensity.Both measurements are normalized against the values obtained insurrounding PBMCs. Vertical dashed line indicate the cutoff value forHMB-45 positive signal. (FIG. 5B) Gallery of 4 HD-CMC detected in twomelanoma patients (patient 30 and 37) using the HD-CMC assay incombination with HMB-45 staining.

FIG. 6 shows the relationship between CMC levels and the overallsurvival of melanoma patients.

FIGS. 7A-7F show DNA copy number variations in single CMCs isolated fromtwo melanoma patients. (FIGS. 7A and 7B) Heatmaps representingchromosomic gains (light grey) and deletions (black) in single CMCs frompatient #30 and patient #37; the hierarchical clustering was performedin R using the heatmap.2 function in the gplots package. Ward's methodwith Manhattan distance metric was used for the clustering. Using mediancentered CNV profiles, cutoff ratios versus the median of 0.675 and 1.7were used to define deletions and amplifications, respectively. Thesecutoffs were used both to color the heatmap and to do the frequencyanalysis (FIGS. 7C and 7D). (FIGS. 7C and 7D) Representative singlePBMCs (top) and CMCs (bottom) DNA CNV profiles. Solid and dashed linesin (FIG. 7D) (bottom) and (FIG. 7F) represents clone A and B,respectively. Adjusted log 10 ratio of read depth of sequencing data areplotted for individual bins (y axis) across genomic regions (x axis).(FIGS. 7E and 7F) Candidate genes located in the amplified and deletedgenomic regions. PMBCs (δ), ‘excluded candidate’ cells (¥) and cellsdisplayed in detail in (FIG. 7C) and (FIG. 7D) ( ). Novel chromosomalamplifications (*).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, in part, on the unexpected discoverythat CMCs can be detected and identified in a biological sample, such asa non-enriched blood sample, by determining the presence or absence of adetectable agent specific for a CMC biomarker and assessing themorphology of the CMCs in the sample. The present disclosure is furtherbased, in part, on the discovery that the presence of CMCs in a samplecan be a diagnostic indicator of metastatic melanoma. As describedherein, CMCs were identified in metastatic melanoma patients.Additionally, despite CMCs having a heterogeneous population in terms ofbiomarker expression and cell morphology within and across melanomapatients, use of a combination of immunofluorescent markers, whichincreased the intensity of the staining of melanoma cell lines, andcertain morphological characteristics of CMCs, provided for a method ofidentifying CMS in a biological sample.

A fundamental aspect of the present disclosure is the robustness of thedisclosed methods. The rare event detection (RED) disclosed herein withregard to CMCs is based on a direct analysis, i.e. non-enriched, of apopulation that encompasses the identification of rare events in thecontext of the surrounding non-rare events. Identification of the rareevents according to the disclosed methods inherently identifies thesurrounding events as non-rare events. Taking into account thesurrounding non-rare events and determining the averages for non-rareevents, for example, average cell size of non-rare events, allows forcalibration of the detection method by removing noise. The result is arobustness of the disclosed methods that cannot be achieved with methodsthat are not based on direct analysis but that instead compare enrichedpopulations with inherently distorted contextual comparisons of rareevents.

Provided herein are methods for identifying CMCs in a biological sampleand for diagnosing subjects with metastatic melanoma. One majoradvantage of the present methods disclosed is the surprisingly highsensitivity with which the methods can classify subjects into a subjectsuffering from metastatic melanoma or a healthy subject, especially innon-enriched blood samples having very low CMC counts. Highclassification sensitivities at low CMC counts facilitates the detectionand diagnosis, and thereby facilitating the timely treatment of asubject. The present disclosure is therefore of particular benefit to asubject who is at an elevated risk of developing melanoma, e.g., due toa genetic predisposition for melanoma, sun exposure, exposure toenvironmental factors, pigmentary characteristics, immunosuppression,family history of melanoma or personal history of melanoma ornon-melanoma skin cancer.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a biomarker” includes a mixture of two or more biomarkers,and the like.

The term “about,” particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

As used in this application, including the appended claims, the singularforms “a,” “an,” and “the” include plural references, unless the contentclearly dictates otherwise, and are used interchangeably with “at leastone” and “one or more.”

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “contains,” “containing,” and any variations thereof, areintended to cover a non-exclusive inclusion, such that a process,method, product-by-process, or composition of matter that comprises,includes, or contains an element or list of elements does not includeonly those elements but can include other elements not expressly listedor inherent to such process, method, product-by-process, or compositionof matter.

The term “biological sample” refers to any specimen from the body of anorganism that can be used for analysis or diagnosis. In the context ofthe present disclosure, a biological sample can be a sample thatcontains or is suspected to contain CMCs. A biological sample obtainedfrom a subject can be any sample that contains or is suspected tocontain cells and encompasses any material in which CMCs can bedetected. For example, a biological sample can include a solid tissuesample (e.g., bone marrow) or a liquid sample (e.g., blood, whole blood,plasma, amniotic fluid, pleural fluid, peritoneal fluid, central spinalfluid, urine, saliva or other body fluid that contains cells). In aparticular aspect of the invention, the biological sample is a bloodsample. As described herein, a preferred sample is a whole blood sample,more preferably a peripheral blood sample. As will be appreciated bythose skilled in the art, a blood sample can include any fraction orcomponent of blood including, without limitation, T-cells, monocytes,neutrophiles, erythrocytes, platelets and microvesicles, such asexosomes and exosome-like vesicles. In the context of this disclosure,blood cells included in a blood sample can encompass any nucleated cellsand are not limited to components of whole blood. As such, blood cellsinclude, for example, both white blood cells (WBCs) as well as rarecells, including CMCs.

The phrase “non-enriched blood sample” refers to a blood sample that hasnot undergone a process so as to substantially add or increase theproportion of target cells or molecules in the sample. The target cellscan be, for example, CMCs in the context of the present disclosure.Accordingly, a non-enriched sample includes a sample that is notenriched for any specific population or subpopulation of nucleatedcells. For example, a non-enriched blood sample may not be enriched forCMCs, WBC, B-cells, T-cells, NK-cells, monocytes, or the like. Theproportion of target cells or molecules can also be relative to anothercomponent in the sample (e.g., WBCs, red blood cells (RBCs), platelets,plasma, or any other molecule known to be present in blood). Severalprocesses that can add or increase the proportion of target cells in asample, including both positive and negative selection methods, areknown in the art. For example, a non-enriched blood sample can include asample that has not undergone: a positive selection for CMCs, such as,for example, enrichment by antibody binding to CMCs or epithelial cells(e.g., EpCAM MicroBeads) or immunomagnetic enrichment (e.g.,magnetic-activated cell sorting (MACs), ferrofluids of coated antibodiesthat bind CMCs, or antibody coated magnetic beads that bind to CMCs); anegative selection, such as, for example, size filtration (e.g., passagethrough an 8 μm filter), depletion of hematopoietic cells by antibodybinding (e.g., anti-CD45 coated magnetic beads) or density gradientcentrifugation; or any combination of both positive and negativeselection. Accordingly, a non-enriched blood sample useful in thedescribed methods can be a sample that has not undergone a positiveand/or negative selection process. As will be appreciated by thoseskilled in the art, a non-enriched blood sample can include a bloodsample that has undergone a process that does not substantially add orincrease the proportion of target cells or molecules in the sample. Suchprocesses can include, for example, addition of a preservative (e.g.,anticoagulant, buffer, adenine, sodium phosphate, citric acid, dextrose,mannitol, sodium chloride), addition of a cyroprotectant (e.g.,glycerol), or lysis of red blood cells (e.g., addition of ammoniumchloride).

The samples of this disclosure can each contain a plurality of cellpopulations and cell subpopulation that are distinguishable by methodswell known in the art (e.g., FACS, immunohistochemistry). For example, ablood sample can contain populations of non-nucleated cells, such aserythrocytes (e.g., 4-5 million/μl) or platelets (150,000-400,000cells/μl), and populations of nucleated cells such as WBCs (e.g.,4,500-10,000 cells/μl) and CMCs (e.g., 1-800 cells/ml). WBCs may containcellular subpopulations of: e.g., neutrophils (2,500-8,000 cells/μl),lymphocytes (1,000-4,000 cells/μl), monocytes (100-700 cells/μl),cosinophils (50-500 cells/μl), basophils (25-100 cells/μl) and the like.

The biological samples of this disclosure may be obtained from anyorganism, including mammals such as humans, primates (e.g., monkeys,chimpanzees, orangutans, and gorillas), cats, dogs, rabbits, farmanimals (e.g., cows, horses, goats, sheep, pigs), and rodents (e.g.,mice, rats, hamsters, and guinea pigs).

It is noted that, as used herein, the terms “subject,” “organism,”“individual” or “patient” are used as synonyms and interchangeably, andrefers to a vertebrate, preferably a mammal. Mammals include, but arenot limited to, humans, primates (e.g., monkeys, chimpanzees,orangutans, and gorillas), cats, dogs, rabbits, farm animals (e.g.,cows, horses, goats, sheep, pigs), and rodents (e.g., mice, rats,hamsters, and guinea pigs).

The subjects of this disclosure include, for example, any subjecthaving: melanoma (e.g., diagnosed with melanoma); suspected of havingmelanoma; suspected of having metastatic melanoma; or being at risk ofdeveloping melanoma. Elevated risks for developing melanoma can be dueto a genetic predisposition for melanoma (e.g., cyclin-dependent kinaseinhibitor 2A (CDKN2A) mutations, mutations in the promoter region of asubunit of telomerase reverse transcriptase (TERT), cyclin-dependentkinase 4 (CDK4) mutations, cyclin-dependent kinase 6 (CDK6) mutations,xeroderma pigmentosum (XP) patients, Cowden syndrome/PTEN hamartomatumor syndrome (PITS) patients; mutations in melanoma susceptibilitylocus on 1p22, alpha melanocyte-stimulating hormone receptor (MCI R)mutations, microphthalmia-associated transcription factor (MITF)mutations, BRCA2 mutations), certain sun exposure (e.g., chronic sunexposure), exposure to environmental factors (e.g., solvents, ionizingradiation, electromagnetic fields, vinyl chloride, polychlorinatedbiphenyls (PCBs)), certain pigmentary characteristics (e.g., low scoreson the Fitzpatrick skin types I-VI), nevi (i.e., birthmarks or beautymarks), immunosuppression, family history of melanoma or personalhistory of melanoma or non-melanoma skin cancer. In some aspects, thesubject is or has been a cancer patient (e.g., melanoma, skin cancer),received an anti-cancer treatment, or discontinued an anti-cancertreatment. Anti-cancer treatments include, for example and withoutlimitation, surgery, drug therapy (e.g., chemotherapy), radiationtherapy, or combinations thereof. In some aspects, the subject istreatment naïve.

The subject can be a healthy organism, including, for example andwithout limitation, an individual or a non-cancer patient in the controlgroup of a clinical study, a cured cancer patient or an individual atrisk of developing cancer.

The subject can also be an animal model for cancer, including, withoutlimitation, a xenograft mouse model, a transgenic mouse carrying atransgenic oncogene, a knockout mouse lacking a proapoptotic gene andothers. A person of skilled in the art understands that many otheranimal models for cancer conditions (e.g., mice or other organisms) arewell known in the art and can be the subject of the methods disclosedherein.

“Melanoma” refers to a form of skin cancer that originates in thepigment-producing cells (melanocytes) of the basal layer of theepidermis. Melanoma can also involve the colored part of the eye or thebowel. As one skilled in the art would understand, melanoma canoriginate in any part of the body that contains melanocytes.

“Metastatic melanoma,” also known as stage IV melanoma, refers to whenmelanoma cells of any kind have spread through the lymph nodes todistant sites in the body and/or to the body's organs. Organs that arefrequently affected by metastatic melanoma include the liver, lungs,bones and brain (Fiddler, Cancer Control. 1995 October; 2(5):398-404).

The “circulating tumor cells” or “CTCs” of this disclosure are tumorcells that are circulating in the bloodstream of an organism.

The “circulating melanoma cells” or “CMCs” of this disclosure aremelanoma cells that are circulating in the bloodstream of an organism.

The term “detectable agent” or “detectable label” refers to a moleculethat can be used for the direct or indirect detection of a biomarker. Awide variety of detectable agents are known in the art and can bereadily identified and used by a person skilled in the art. Suitabledetectable agents include, but are not limited to, fluorescent dycs(e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™,rhodamine, Texas Red, tetrarhodamine isothiocynate (TRITC), Cy3, Cy5,Alexa Fluor® 647, Alexa Fluor® 555, Alexa Fluor® 488), fluorescentprotein markers (e.g., green fluorescent protein (GFP), phycoerythrin,etc.), enzymes (e.g., luciferase, horseradish peroxidase, alkalinephosphatase, etc.), nanoparticles, biotin, digoxigenin, metals, and thelike.

The term “immunofluorescent marker” refers to a detectable agent that isan antibody or functional fragment thereof that targets a fluorescentdye to a specific molecule within or on a cell. An immunofluorescentmarker can be used in methods that employ a fluorescent light microscopeto produce immunostaining for a desired sample. An immunofluorescentmarker can also be employed in immunocytochemistry (ICC) orimmunohistochemistry (IHC) methods described herein. In the context ofthe present disclosure, an immunofluorescent marker can be used todetect a CMC as described herein.

The term “antibody” refers to any immunoglobulin or derivative thereof,whether natural or wholly or partially synthetically produced. Allantibody derivatives which maintain specific binding ability can also beused in the disclosed methods. The antibodies of this disclosure canbind specifically to a biomarker. For example, the antibodies can bindspecifically to a single biomarker (e.g., chondroitin sulfateproteoglycan 4 (CSPG4)). Additionally, the antibodies can bepan-specific. For sample, pan-specific antibodies of this disclosure canbind specifically to one or more members of a biomarker family (e.g.,one or more members of the chondroitin sulfate proteoglycan family,including chondroitin sulfate proteoglycan 1, 2, 3, 4, 5, 6, 7 and 8).The antibody can have a binding domain that is homologous or largelyhomologous to an immunoglobulin binding domain and can be derived fromnatural sources, or partly or wholly synthetically produced. Theantibody can be a monoclonal or polyclonal antibody. In some aspects,the antibody is a single-chain antibody. In some aspects, the antibodyincludes a single-chain antibody fragment. In some aspects, the antibodycan be an antibody fragment including, but not limited to, Fab, Fab′,F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments. Due to their smallersize antibody fragments can offer advantages over intact antibodies incertain applications. Alternatively or additionally, the antibody cancomprise multiple chains which are linked together, for example, bydisulfide linkages, and any functional fragments obtained from suchmolecules, wherein such fragments retain specific-binding properties ofthe parent antibody molecule. Those of skill in the art will appreciatethat the antibody can be provided in any of a variety of formsincluding, for example, humanized, partially humanized, chimeric,chimeric humanized, etc. The antibody can be prepared using any suitablemethods known in the art. For example, the antibody can be enzymaticallyor chemically produced by fragmentation of an intact antibody or it canbe recombinantly produced from a gene encoding the partial antibodysequence.

The term “biomarker” refers to a biological molecule, or a fragment of abiological molecule, the change and/or the detection of which can becorrelated with a particular physical condition or state of a CMC. Theterms “marker” and “biomarker” are used interchangeably throughout thedisclosure. Such biomarkers include, but are not limited to, biologicalmolecules comprising nucleotides, nucleic acids, nucleosides, aminoacids, sugars, fatty acids, steroids, metabolites, peptides,polypeptides, proteins, carbohydrates, lipids, hormones, antibodies,regions of interest that serve as surrogates for biologicalmacromolecules and combinations thereof (e.g., glycoproteins,ribonucleoproteins, lipoproteins). The term also encompasses portions orfragments of a biological molecule, for example, peptide fragment of aprotein or polypeptide. In the context of the present disclosure,exemplary biomarkers for CMCs include chondroitin sulfate proteoglycan 4(CSPG4), premelanosome protein (Pmel17) and S100 calcium-binding proteinA1 (S100A1).

“Chondroitin sulfate proteoglycan 4” or “CSPG4,” also known as highmolecular weight melanoma associated antigen (HMW-MAA) and melanomachondroitin sulfate proteoglycan (MCSP), is a membrane-boundproteoglycan that mediates both cell-cell and cell-extracellular matrixinteractions and has been associated with the metastatic potential ofmelanoma cells (Price et al., Pigment Cell Melanoma Res. 2011 December;24(6):1148-57; Yang et al., J Cell Biol. 2004 Jun. 21; 165(6):881-91;Yang et al., Cancer Res. 2009 Oct. 1; 69(19):7538-47; Iida et al., JBiol Chem. 2001 Jun. 1:276(22):18786-94).

“Premelanosome protein” or “Pmel17,” also know as Silver, SILV, GP100and ME20, refers to is a 100 kDa type I transmembrane glycoprotein thatis expressed primarily in pigment cells of the skin and eye and isresponsible for the formation of fibrillar sheets within the pigmentorganelle, the melanosome (Kim et al., Pigment Cell Res. 1996 February;9(1):42-8; Watt et al., Pigment Cell Melanoma Res. 2013 May;26(3):300-15). HMB-45 is a monoclonal antibody that specifically reactsagainst Pmel 17, and stands for Human Melanoma Black (Gown et al., Am JPathol. 1986 May; 123(2):195-203). HMB-45 can be used in anatomicpathology as a marker for melanoma (Mahnood et al., Mod Pathol. 2002December; 15(12):1288-93).

“S100 calcium-binding protein A1” or “S100A1” refers to a member of theS100 family of proteins containing four EF-hand calcium-binding motifsin its dimerized form, which in humans is encoded by the S100A1 gene(Marenholz et al., Biochem Biophys Res Commun. 2004 Oct. 1;322(4):1111-22; Morii et al., Biochem Biophys Res Commun. 1991 Feb. 28;175(1):185-91). S100 proteins are localized in the cytoplasm and/ornucleus of a wide range of cells, and involved in the regulation of anumber of cellular processes such as cell cycle progression anddifferentiation. S100A1 may function in stimulation of Ca2+-induced Ca2+release, inhibition of microtubule assembly, and inhibition of proteinkinase C-mediated phosphorylation. S100A1 expression has been seen inmalignant melanomas in a diffuse reaction (Nonaka et al., J CutanPathol. 2008 November; 35(11):1014-9).

“Morphology” or “morphological characteristic.” when used in referenceCMCs, refers to a feature, form or structure of CMCs that is sharedbetween CMCs. Examples of such features, forms or structures include,without limitation, the presence of an intact nucleus, the nucleus size,the nucleus shape, the cell size, the cell shape and the nuclear tocytoplasmic ratio. Accordingly, in some aspects, for example, themorphology indicative of a CMC is a nuclear to cytoplasmic ratio of lessthan 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, or 1.0.

As used herein, the term “cluster” means two or more CMCs with touchingcell membranes.

“Nucleic acid specific stain” refers to a molecule that selectivelybinds to a nucleic acid in a sample and produces a distinctivedetectable signal, either directly or indirectly. A nucleic acidspecific stain includes, but is not limited to, a molecule that binds todouble stranded deoxyribonucleic acids (DNA) via intercalation, majorgroove binding, minor groove binding, external binding orbis-intercalation. Examples of intercalating molecules include ethidiumbromide and propidium iodide. Examples of minor-groove binders include4′,6-diamidino-2-phenylindole (DAPI) and bis-benzimides dyes (also knownas Hoechst dyes) (e.g., Hoechst 33258. Hoechst 33342, and Hoechst34580). Examples of other nucleic acid stains include acridine orange,7-aminoactinomycin D (7-AAD), SYTOX Blue, Chromomycin A3, Mithramycin,YOYO-1, SYTOX Green, TOTO-1, TO-PRO-1, TO-PRO: Cyanine Monomer, ThiazoleOrange, CyTRAK Orange, LDS 751, SYTOX Orange, TOTO-3, TO-PRO-3, DRAQ5,and DRAQ7.

“Automated fluorescent microscopy” refers to a system of operatingand/or controlling an optical microscope that uses fluorescence orphosphorescence by automatic devises to generate an image of a samplethereby reducing human intervention to a minimum. Such a system caninclude capturing images of the sample and analyzing the images toidentify the presence or absence of CMC's in the sample.

As used herein, the term “predetermined number” when used in referenceto the amount of CMCs relative to the amount of another compound orsample volume is intended to mean a number of CMCs that is establishedin advance of performing a method described herein that is indicative ofa subject having metastatic melanoma. The predetermined number can beidentified through prior experimental observations. In the context ofthe present disclosure, a predetermined number of CMCs present in asample that is at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10, 20,50, 100, 200, 300, 400 or 500 CMCs per ml of sample can be indicative ofa subject having metastatic melanoma. In another aspect, a predeterminednumber of CMCs indicative of a subject having metastatic melanoma can bethe number of CMCs per another component of the sample, such as, but notlimited to, WBCs. The number of WBCs in the blood of a normal individualcan be between about 4.0×10⁹ to about 12×10⁹ WBCs per liter of blooddepending upon a number of factors including age, gender, and ethnicity,with the average individual after age 11 years having about 6.0-7.5×10⁹WBCs per liter (Vital and Health Statistics, Series 11, No. 247 (March2005). Accordingly, in some aspects, the ratio of CMCs per WBCs in thesample can be indicative of a subject having metastatic melanoma. Forexample, the number of CMCs per WBCS can be 0.5, 1.0, 1.5, 2.0, 2.5,3.0, 4.0, 5.0, 10, 20, 50, 100, 200, 300, 400 or 500 CMCs per6.0-7.5×10⁶ WBCs.

In some embodiments, the disclosure provides a method for identifyingCMCs in a biological sample. The method can include the steps of: (a)contacting a biological sample of non-enriched blood with one or moredetectable agents, wherein at least one of the one or more detectableagents is specific for a biomarker of CMCs; (b) determining the presenceor absence of the one or more detectable agents in or on nucleated cellsin the sample; and (c) assessing the morphology of the nucleated cellshaving the one or more detectable agents, wherein the CMCs areidentified based on a combination of the presence or absence of the oneor more detectable agents and morphological characteristics of thenucleated cells.

In some embodiments, the disclosure provides a method for diagnosingmetastatic melanoma. In one aspect, the method for diagnosing metastaticmelanoma can include the steps of: (a) contacting a biological sample ofnon-enriched blood with one or more detectable agents, wherein thesample was obtained from a subject suspected of having metastaticmelanoma or diagnosed with having melanoma, wherein at least one of theone or more detectable agents is specific for a biomarker of CMCs; (b)determining the presence or absence of the one or more detectable agentsin or on nucleated cells present in the sample; (c) assessing themorphology of the nucleated cells having the one or more detectableagents; and (d) identifying the presence of CMCs in the sample based ona combination of the presence or absence of the one or more detectableagents and morphological characteristics of the nucleated cells, whereinthe subject is diagnosed with metastatic melanoma when a predeterminednumber of CMCs is present in the sample.

In some embodiments of the disclosure, one or more detectable agents canbe used in the methods disclosed herein. For example, in some aspects,two, three, four, five, six, seven, eight, nine, ten or more detectableagents can be used in the claimed methods. In some aspects, twodetectable agents can be used in the claimed methods. In some aspects,three detectable agents can be used in the claimed methods. In someaspects, four detectable agents can be used in the claimed methods. Insome aspects, five detectable agents can be used in the claimed methods.In some aspects, six detectable agents can be used in the claimedmethods. In some aspects, seven detectable agents can be used in theclaimed methods. In some aspects, eight detectable agents can be used inthe claimed methods. In some aspects, nine detectable agents can be usedin the claimed methods. In some aspects, ten or more detectable agentscan be used in the claimed methods. As will be understood by a personskilled in the art, when there are two or more agents used in a methodof the disclosure the selection of the detectable agents can bedependent upon specific features and/or characteristics of theindividual detectable agents. For example, in some aspects, if two ormore of the detectable agents are detectable by fluorescence, thespecific excitation wavelength and/or emission wavelength of thefluorophores for each detectable agent will not overlap. Accordingly, aperson skilled in the art would be able to readily ascertain whichcombination of detectable agents can be used in combination for thedisclosed methods.

In some aspects, the one or more detectable agents used in the methodscan be an immunofluorescent marker, and in particular embodiments, theimmunofluorescent marker specifically binds a biomarker of CMCs. Anexample of such an immunofluorescent marker includes, but is not limitedto, an antibody or functional fragment thereof that specifically bindsto CSPG4, Pmel17 or S100A1. In some aspects, the antibody is monoclonal.In some aspects, the one or more detectable agents can include two,three, four, five, six, seven or more immunofluorescent markers. Forexample, as disclosed herein, the use of multiple immunofluorescentmarkers, even directed to the same biomarker, can increase thesensitivity of the methods disclosed. According, in some aspects, theone or more detectable agents include two immunofluorescent markers. Insome aspects, the one or more detectable agents include threeimmunofluorescent markers. In some aspects, the one or more detectableagents include four immunofluorescent markers. In some aspects, the oneor more detectable agents include five immunofluorescent markers. Insome aspects, the one or more detectable agents include siximmunofluorescent markers. In some aspects, the one or more detectableagents include seven or more immunofluorescent markers. As will beunderstood by a person skilled in the art, when multipleimmunofluorescent markers that target the same biomarker are used in themethods disclosed, it may be desirable for the markers to specificallyrecognize distinct and/or distant epitopes of the target biomarker.Accordingly, selection of immunofluorescent markers that arecomplementary to each other (e.g., do not competitively inhibit bindingto the target biomarker) can be selected by a person skilled in the art.

In some embodiments of the disclosure, the one or more detectable agentsused in the disclosed method is a nucleic acid specific stain. Forexample, in some aspects, the nucleic acid specific stain is a Hoechststain or any other nucleic acid specific stain disclosed herein or wellknown in the art. As will be appreciated by a person skilled in the art,the selection of a nucleic acid specific stain can be dependent uponspecific features and/or characteristics of the other detectable agentsused in the disclosed method. A person skilled in the art could readilyascertain which nucleic acid specific stain would be compatible with theother detectable agents.

In some embodiments of the disclosure, the one or more detectable agentscomprise an immunofluorescent marker specific for a component of a bloodsample other than the CMCs. For example, in some aspects, theimmunofluorescent marker is specific for WBCs. Such an WBC specificmaker can be an antibody specific for cluster of differentiation 45(CD45).

In some embodiments of the disclosure, the one or more detectable agentsused in the disclosed methods can identify a CMC present in the sampleeven in the presence of other nucleated cells. For example, in someaspects, the immunofluorescent staining of CMCs is negative for anantibody or functional fragment thereof that specifically binds to CD45(i.e., CD45 (−)). However, the surrounding WBCs can be CD45 (+). In someaspects, the immunofluorescent staining of CMCs is positive for Hoeschststaining (i.e., Hoechst stain (+)). In some aspects, theimmunofluorescent staining of CMCs is positive for CSPG4 (i.e., CSPG4(+)). Accordingly, in particular embodiments, all nucleated cells areretained and immunofluorescently stained with one or more antibodiestargeting a CMC biomarker (e.g., CSPG4, Pmel17 or S100A1), an antibodytargeting the common leukocyte antigen CD45, and a nucleic acid specificstain (e.g., Hoechst stain). The nucleated blood cells can be imaged inmultiple fluorescent channels to produce high quality and highresolution digital images that retain fine cytologic details of nuclearcontour and cytoplasmic distribution. While the surrounding WBCs can beidentified with the antibody targeting CD45, the CMCs can be identifiedas, for example, CSPG4 (+), Hoechst stain (+) and CD45 (−). Accordingly,in the methods described herein, the CMCs can comprise distinctimmunofluorescent staining from surrounding nucleated cells.

In some embodiments of the disclosure, the immunofluorescent staining ofCMCs is positive for an antibody or functional fragment thereof thatspecifically binds to a CMC biomarker (e.g., CSPG4, Pmel17 or S100A1)based on a predetermine threshold intensity of fluorescence upon whichto classify a candidate cell as being positive for CSPG4. For example,identifying a CMC as being positive for CSPG4 can be a candidate cellhaving a standard deviation of the mean (SDOM) of greater than or equalto 2. Accordingly, in some aspects, the immunofluorescent staining ofCMCs is positive for an antibody or functional fragment thatspecifically binds to CSPG4 or another CMC biomarker and is detectableat an SDOM of greater than or equal to 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7,8, 9 or 10. In some aspects, the immunofluorescent staining of CMCs isdetectable at an SDOM of greater than or equal to 2. In some aspects,the immunofluorescent staining of CMCs is detectable at an SDOM ofgreater than or equal to 2.5. In some aspects, the immunofluorescentstaining of CMCs is detectable at an SDOM of greater than or equal to 3.In some aspects, the immunofluorescent staining of CMCs is detectable atan SDOM of greater than or equal to 3.5. In some aspects, theimmunofluorescent staining of CMCs is detectable at an SDOM of greaterthan or equal to 4. In some aspects, the immunofluorescent staining ofCMCs is detectable at an SDOM of greater than or equal to 4.5. In someaspects, the immunofluorescent staining of CMCs is detectable at an SDOMof greater than or equal to 5. In some aspects, the immunofluorescentstaining of CMCs is detectable at an SDOM of greater than or equal to 6.In some aspects, the immunofluorescent staining of CMCs is detectable atan SDOM of greater than or equal to 7. In some aspects, theimmunofluorescent staining of CMCs is detectable at an SDOM of greaterthan or equal to 8. In some aspects, the immunofluorescent staining ofCMCs is detectable at an SDOM of greater than or equal to 9. In someaspects, the immunofluorescent staining of CMCs is detectable at an SDOMof greater than or equal to 10.

In some embodiments, the presence or absence of a detectable agent,including an immunofluorescent marker, in or on nucleated cells, such asCMCs or WBCs, can result in distinct immunofluorescent stainingpatterns. Using the detectable agents disclosed herein, the methods ofthe disclosure can identify CMCs is a biological sample by determiningthe presence or absence of the one or more detectable agents on or inthe candidate cells by comparing distinct immunofluorescent staining ofCMCs with distinct immunofluorescent staining of WBCs. Immunofluorescentstaining patterns for CMCs and WBCs may differ based on which markersare detected in the respective cells. In some embodiments, determiningpresence or absence of one or more immunofluorescent markers includescomparing the distinct immunofluorescent staining of CMCs with thedistinct immunofluorescent staining of WBCs using, for example,immunofluorescent staining of CD45, which distinctly identifies WBCs.There are other detectable markers or combinations of detectable markersthat bind to the various subpopulations of WBCs. These may be used invarious combinations, including in combination with or as an alternativeto immunofluorescent staining of CD45.

In some embodiments, the method further includes analyzing the nucleatedcells by nuclear detail, nuclear contour, presence or absence ofnucleoli, quality of cytoplasm, quantity of cytoplasm, intensity ofimmunofluorescent staining patterns. A person of skilled in the artunderstands that the morphological characteristics of this disclosuremay include any feature, property, characteristic, or aspect of a cellthat can be determined and correlated with the detection of a CMC.

In some embodiments of the disclosure, the methods include assessing themorphology of the nucleated cells having the one or more detectableagents as described herein (e.g., cells that are CSPG4 (+), Hoechststain (+) and CD45 (−)). Assessing the morphology of the cells in thebiological sample can include, in some aspects, comparing themorphological characteristics of CMCs with the morphologicalcharacteristics of surrounding WBCs. For example, the morphologicalcharacteristics that are compared can include nucleus size, nucleusshape, cell size, cell shape, and/or nuclear to cytoplasmic ratio. Insome aspects of the disclosure, a nuclear to cytoplasmic ratio of lessthan 5.0, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 or 1.0 can indicate the presenceof a CMC. In some aspects, the nuclear to cytoplasmic ratio indicatingthe presence of a CMC is less than 5.0. In some aspects, the nuclear tocytoplasmic ratio indicating the presence of a CMC is less than 4.0. Insome aspects, the nuclear to cytoplasmic ratio indicating the presenceof a CMC is less than 3.5. In some aspects, the nuclear to cytoplasmicratio indicating the presence of a CMC is less than 3.0. In someaspects, the nuclear to cytoplasmic ratio indicating the presence of aCMC is less than 2.5. In some aspects, the nuclear to cytoplasmic ratioindicating the presence of a CMC is less than 2.0. In some aspects, thenuclear to cytoplasmic ratio indicating the presence of a CMC is lessthan 1.5. In some aspects, the nuclear to cytoplasmic ratio indicatingthe presence of a CMC is less than 1.0.

In some embodiments, the methods of the disclosure include detection ofhigh definition CMCs (HD-CMCs). HD-CMCs can be, in some aspect, CSPG4(+) with an SDOM of greater than or equal to 2, Hoechst stain (+). CD45(−), and have a morphologically distinct feature from surrounding WBCsincluding having an intact nucleus with a nuclear to cytoplasmic ratioof less than 2.5. CSPG4 (+), Hoechst stain (+) and CD45 (−) intensitiescan be categorized as measurable features during HD-CTC enumeration asdescribed herein and/or as described in Nieva et al., Phys Biol 9:016004(2012). The enrichment-free, direct analysis employed by the methodsdisclosed herein results in high sensitivity and high specificity, whileadding high definition cytomorphology to enable detailed morphologiccharacterization of a CMC population, including a population that isheterogenous as described herein.

While CMCs can be identified as being CSPG4 (+), Hoechst stain (+) andCD45 (−) cells, the methods of the invention can be practiced with anyother biomarkers that one of skill in the art selects for generating CMCdata and/or identifying CMCs. One skilled in the art knows how to selecta morphological feature, biological molecule, or a fragment of abiological molecule, the change and/or the detection of which can becorrelated with a CMC.

CMCs, which can be present as single cells or in clusters of CMCs, canbe cells shed from solid melanoma tumors and be present in very lowconcentrations in the circulation of subjects. Accordingly, detection ofCMCs in a blood sample can be referred to as rare event detection. CMCscan have an abundance of less than 1:1,000 in a blood cell population,e.g., an abundance of less than 1:5,000, 1:10,000, 1:30.000, 1:50:000,1:100,000, 1:300,000, 1:500,000, or 1:1,000,000. In some embodiments,the CMC has an abundance of 1:50:000 to 1:100,000 in the cellpopulation.

The samples of this disclosure may be obtained by any means, including,e.g., by means of solid tissue biopsy or fluid biopsy (see, e.g.,Marrinucci D. et al., 2012, Phys. Biol. 9 016003). A blood sample may beextracted from any source known to include blood cells or componentsthereof, such as venous, arterial, peripheral, tissue, cord, and thelike. The samples may be processed using well known and routine clinicalmethods (e.g., procedures for drawing and processing whole blood). Insome embodiments, a blood sample is drawn into anti-coagulent bloodcollection tubes (BCT), which may contain EDTA or Streck Cell-Free DNA™.In other embodiments, a blood sample is drawn into CellSave® tubes(Veridex). A blood sample may further be stored for up to 12 hours, 24hours, 36 hours, 48 hours, or 60 hours before further processing.

In some embodiments, the methods of this disclosure comprise an initialstep of obtaining a WBC count for the blood sample. In certainembodiments, the WBC count may be obtained by using a HemoCue® WBCdevice (Hemocue, Ängelholm, Sweden). In some embodiments, the WBC countis used to determine the amount of blood required to plate a consistentloading volume of nucleated cells per slide and to calculate back theequivalent of CMCs per blood volume.

In some embodiments, the methods of this disclosure comprise an initialstep of lysing erythrocytes in the blood sample. In some embodiments,the erythrocytes are lysed, e.g., by adding an ammonium chloridesolution to the blood sample. In certain embodiments, a blood sample issubjected to centrifugation following erythrocyte lysis and nucleatedcells are resuspended, e.g., in a PBS solution.

In some embodiments, nucleated cells from a sample, such as a bloodsample, are deposited as a monolayer on a planar support. The planarsupport may be of any material, e.g., any fluorescently clear material,any material conducive to cell attachment, any material conducive to theeasy removal of cell debris, any material having a thickness of <100 μm.In some embodiments, the material is a film. In some embodiments thematerial is a glass slide. In certain embodiments, the methodencompasses an initial step of depositing nucleated cells from the bloodsample as a monolayer on a glass slide. The glass slide can be coated toallow maximal retention of live cells (See, e.g., Marrinucci D. et al.,2012, Phys. Biol. 9 016003). In some embodiments, about 0.5 million, 1million, 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4million, 4.5 million, or 5 million nucleated cells are deposited ontothe glass slide. In some embodiments, the methods of this disclosurecomprise depositing about 3 million cells onto a glass slide. Inadditional embodiments, the methods of this disclosure comprisedepositing between about 2 million and about 3 million cells onto theglass slide. In some embodiments, the glass slide and immobilizedcellular samples are available for further processing or experimentationafter the methods of this disclosure have been completed.

In some embodiments, the methods of the disclosure, includingdetermining the presence or absence of one or more detectable agents inor on nucleated cells in the sample and/or assessing the morphology ofthe nucleated cells having the one or more detectable agents can beperformed by automated fluorescent microscopy. For example, fluorescentscanning microscopy to detect immunofluorescent staining of nucleatedcells in a blood sample has been described by Marrinucci D. et al.,2012, Phys. Biol. 9 016003, and can be used with the disclosed methods.However, a person skilled in the art will appreciate that a number ofmethods can be used to identify CMCs in a biological sample, includingmicroscopy based approaches, mass spectrometry approaches, such asMS/MS. LC-MS/MS, multiple reaction monitoring (MRM) or SRM andproduct-ion monitoring (PIM) and also including antibody based methodssuch as immunofluorescence, immunohistochemistry, immunoassays such asWestern blots, enzyme-linked immunosorbant assay (ELISA),immunoprecipitation, radioimmunoassay, dot blotting, and FACS.Immunoassay techniques and protocols are generally known to thoseskilled in the art (Price and Newman, Principles and Practice ofImmunoassay, 2nd Edition, Grove's Dictionaries, 1997; and Gosling,Immunoassays: A Practical Approach, Oxford University Press, 2000.) Avariety of immunoassay techniques, including competitive andnon-competitive immunoassays, can be used (Self et al., Curr. Opin.Biotechnol., 7:60-65 (1996), see also John R. Crowther, The ELISAGuidebook, 1st ed., Humana Press 2000, ISBN 0896037282 and, AnIntroduction to Radioimmunoassay and Related Techniques, by Chard T,ed., Elsevier Science 1995, ISBN 0444821198).

A person of skill in the art will further appreciate that the presenceor absence of biomarkers may be detected using any class ofmarker-specific binding reagents known in the art, including, e.g.,antibodies, aptamers, fusion proteins, such as fusion proteins includingprotein receptor or protein ligand components, or biomarker-specificsmall molecule binders. In some embodiments, the presence or absence ofCSPGT4, PmeL17 or S100A1 is determined by an antibody.

The antibodies of this disclosure can bind specifically to a biomarker.The antibody can be prepared using any suitable methods known in theart. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, Antibodies: A Laboratory Manual (1988); Goding, MonoclonalAntibodies: Principles and Practice (2d ed. 1986). The antibody can beany immunoglobulin or derivative thereof, whether natural or wholly orpartially synthetically produced. All derivatives thereof which maintainspecific binding ability can also be used. The antibody can have abinding domain that is homologous or largely homologous to animmunoglobulin binding domain and can be derived from natural sources,or partly or wholly synthetically produced. The antibody can be amonoclonal or polyclonal antibody. In some embodiments, an antibody is asingle chain antibody. Those of skill in the art will appreciate thatantibody can be provided in any of a variety of forms including, forexample, humanized, partially humanized, chimeric, chimeric humanized,etc. The antibody can be an antibody fragment including, but not limitedto, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments. Theantibody can be produced by any means. For example, the antibody can beenzymatically or chemically produced by fragmentation of an intactantibody and/or it can be recombinantly produced from a gene encodingthe partial antibody sequence. The antibody can comprise a single chainantibody fragment. Alternatively or additionally, the antibody cancomprise multiple chains which are linked together, for example, bydisulfide linkages, and any functional fragments obtained from suchmolecules, wherein such fragments retain specific-binding properties ofthe parent antibody molecule. Because of their smaller size asfunctional components of the whole molecule, antibody fragments canoffer advantages over intact antibodies for use in certainimmunochemical techniques and experimental applications.

One or more detectable agents can be used in the methods describedherein for direct or indirect detection of the biomarkers whenidentifying CMCs in the methods of the disclosure. A wide variety ofdetectable labels can be used, with the choice of label depending on thesensitivity required, ease of conjugation with the antibody, stabilityrequirements, and available instrumentation and disposal provisions.Those skilled in the art are familiar with selection of a suitabledetectable label based on the assay detection of the biomarkers in themethods of the invention. Suitable detectable labels include, but arenot limited to, fluorescent dyes (e.g., fluorescein, fluoresceinisothiocyanate (FITC), Oregon Green™, rhodamine, Texas red,tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, Alexa Fluor® 647, AlexaFluor® 555, Alexa Fluor® 488), fluorescent markers (e.g., greenfluorescent protein (GFP), phycoerythrin, etc.), enzymes (e.g.,luciferase, horseradish peroxidase, alkaline phosphatase, etc.),nanoparticles, biotin, digoxigenin, metals, and the like.

For mass-spectrometry based analysis, differential tagging with isotopicreagents, e.g., isotope-coded affinity tags (ICAT) or the more recentvariation that uses isobaric tagging reagents, iTRAQ (AppliedBiosystems, Foster City, Calif.), followed by multidimensional liquidchromatography (LC) and tandem mass spectrometry (MS/MS) analysis canprovide a further methodology in practicing the methods of thisdisclosure.

A chemiluminescence assay using a chemiluminescent antibody can be usedfor sensitive, non-radioactive detection of proteins. An antibodylabeled with fluorochrome can also be suitable. Examples offluorochromes include, without limitation, DAPI, fluorescein. Hoechst33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texasred, and lissamine. Indirect labels include various enzymes well knownin the art, such as horseradish peroxidase (HRP), alkaline phosphatase(AP), beta-galactosidase, urease, and the like. Detection systems usingsuitable substrates for horseradish-peroxidase, alkaline phosphatase,beta.-galactosidase are well known in the art.

A signal from the direct or indirect label can be analyzed, for example,using a microscope, such as a fluorescence microscope or a fluorescencescanning microscope. Alternatively, a spectrophotometer can be used todetect color from a chromogenic substrate; a radiation counter to detectradiation such as a gamma counter for detection of ¹²⁵I; or afluorometer to detect fluorescence in the presence of light of a certainwavelength. If desired, assays used to practice the methods of thisdisclosure can be automated or performed robotically, and the signalfrom multiple samples can be detected simultaneously.

CMCs can be detected with any microscopic method known in the art. Insome embodiments, the method is performed by fluorescent scanningmicroscopy. In certain embodiments the microscopic method provideshigh-resolution images of CMCs and their surrounding WBCs (see, e.g.,Marrinucci D. et al., 2012, Phys. Biol. 9 016003)). In some embodiments,a slide coated with a monolayer of nucleated cells from a sample, suchas a non-enriched blood sample, is scanned by a fluorescent scanningmicroscope and the fluorescence intensities from immunofluorescentmarkers and nuclear stains are recorded to allow for the determinationof the presence or absence of each immunofluorescent marker and theassessment of the morphology of the nucleated cells. In someembodiments, microscopic data collection and analysis is conducted in anautomated manner.

In some embodiments, identifying CMCs includes detecting one or morebiomarkers, for example, CSPG4, Pmel17, S100A1 or CD45. A biomarker isconsidered “present” in a cell if it is detectable above the backgroundnoise of the respective detection method used (e.g., 2-fold, 3-fold,5-fold, or 10-fold higher than the background; e.g., 2a or 3a overbackground). In some embodiments, a biomarker is considered “absent” ifit is not detectable above the background noise of the detection methodused (e.g., <1.5-fold or <2.0-fold higher than the background signal;e.g., <1.5a or <2.0a over background).

In some embodiments, the presence or absence of immunofluorescentmarkers in nucleated cells is determined by selecting the exposure timesduring the fluorescence scanning process such that all immunofluorescentmarkers achieve a pre-set level of fluorescence on the WBCs in the fieldof view. Under these conditions, CMC-specific immunofluorescent markers,even though absent on WBCs are visible in the WBCs as background signalswith fixed heights. Moreover, WBC-specific immunofluorescent markersthat are absent on CMCs are visible in the CMCs as background signalswith fixed heights. In some aspects, a cell is considered positive foran immunofluorescent marker (i.e., the marker is considered present) ifits fluorescent signal for the respective marker is significantly higherthan the fixed background signal (e.g., 2-fold, 3-fold, 5-fold, or10-fold higher than the background; e.g., 2σ or 3σ over background). Forexample, a nucleated cell can be considered CD45 (+) if its fluorescentsignal for CD45 is significantly higher than the background signal. Acell is considered negative for an immunofluorescent marker (i.e., themarker is considered absent) if the cell's fluorescence signal for therespective marker is not significantly above the background signal(e.g., <1.5-fold or <2.0-fold higher than the background signal; e.g.,<1.5σ or <2.0σ over background).

Typically, each microscopic field contains both CMCs and WBCs. Incertain embodiments, the microscopic field shows at least 1, 5, 10, 20,50, or 100 CMCs. In certain embodiments, the microscopic field shows atleast 10, 25, 50, 100, 250, 500, or 1,000 fold more WBCs than CMCs. Incertain embodiments, the microscopic field comprises one or more CMCs orCMC clusters surrounded by at least 10, 50, 100, 150, 200, 250, 500,1,000 or more WBCs.

In some embodiments of the methods for diagnosing, the disclosed methodcan include enumeration of CMCs that are present in the blood sample. Insome embodiments, a positive diagnosis of metastatic melanoma comprisesdetection of at least 0.5 CMC/ml of blood, 1.0 CMC/mL of blood, 1.5CMCs/mL of blood, 2.0 CMC/mL of blood, 2.5 CMCs/mL of blood, 3.0 CMCs/mLof blood, 3.5 CMCs/mL of blood, 4.0 CMCs/mL of blood, 4.5 CMCs/mL ofblood, 5.0 CMCs/mL of blood, 5.5 CMCs/mL of blood, 6.0 CMCs/mL of blood,6.5 CMCs/mL of blood, 7.0 CMCs/mL of blood, 7.5 CMCs/mL of blood, 8.0CMCs/mL of blood, 8.5 CMCs/mL of blood, 9.0 CMCs/mL of blood, 9.5CMCs/mL of blood, 10 CMCs/mL of blood, 20 CMCs/mL of blood, 30 CMCs/mLof blood, 40 CMCs/mL of blood, 50 CMCs/mL of blood, 60 CMCs/mL of blood,70 CMCs/mL of blood, 80 CMCs/mL of blood, 90 CMCs/mL of blood, 100CMCs/mL of blood, 200 CMCs/mL, 300 CMCs/mL, 400 CMCs/mL, 500 CMCs/mL ormore. In a particular embodiment, a positive diagnosis of metastaticmelanoma includes detection of at least 0.5 CMC/mL of blood. In aparticular embodiment, a positive diagnosis of metastatic melanomaincludes detection of at least 1 CMC/mL of blood. In a particularembodiment, a positive diagnosis of metastatic melanoma includesdetection of at least 2 CMC/mL of blood. In a particular embodiment, apositive diagnosis of metastatic melanoma includes detection of at least5 CMC/mL of blood. In a particular embodiment, a positive diagnosis ofmetastatic melanoma includes detection of at least 10 CMC/mL of blood.In a particular embodiment, a positive diagnosis of metastatic melanomaincludes detection of at least 20 CMC/mL of blood. In a particularembodiment, a positive diagnosis of metastatic melanoma includesdetection of at least 50 CMC/mL of blood. In a particular embodiment, apositive diagnosis of metastatic melanoma includes detection of at least100 CMC/mL of blood. In a particular embodiment, a positive diagnosis ofmetastatic melanoma includes detection of at least 500 CMC/mL of blood.

In some embodiments of the methods for diagnosing, the disclosed methodcan include detecting CMC clusters. In some embodiments, a positivediagnosis of metastatic melanoma includes detection of at least 0.1 CMCcluster/mL of blood, 0.2 CMC clusters/mL of blood, 0.3 CMC clusters/mLof blood, 0.4 CMC clusters/mL of blood, 0.5 CMC clusters/mL of blood,0.6 CMC clusters/mL of blood, 0.7 CMC clusters/mL of blood, 0.8 CMCclusters/mL of blood, 0.9 CMC clusters/mL of blood, 1 CMC cluster/mL ofblood, 2 CMC clusters/mL of blood, 3 CMC clusters/mL of blood, 4 CMCclusters/mL of blood, 5 CMC clusters/mL, of blood, 6 CMC clusters/ml. ofblood, 7 CMC clusters/mL of blood, 8 CMC clusters/mL of blood, 9 CMCclusters/mL of blood, 10 clusters/mL or more. In a particularembodiment, a positive diagnosis of metastatic melanoma comprisesdetection of at least 0.1 CMC cluster/mL of blood. In a particularembodiment, a positive diagnosis of metastatic melanoma comprisesdetection of at least 0.5 CMC cluster/mL of blood. In a particularembodiment, a positive diagnosis of metastatic melanoma comprisesdetection of at least 1 CMC cluster/mL of blood. In a particularembodiment, a positive diagnosis of metastatic melanoma comprisesdetection of at least 2 CMC cluster/mL of blood. In a particularembodiment, a positive diagnosis of metastatic melanoma comprisesdetection of at least 10 CMC cluster/mL of blood.

In some embodiments, the method disclosed herein for diagnosingmetastatic melanoma in a subject has a specificityof >60%, >70%, >80%, >90% or higher. in additional embodiments, themethod for diagnosing metastatic melanoma in a subject has aspecificity >60% at a classification threshold of 0.5 CMCs/mL of blood.In additional embodiments, the method for diagnosing metastatic melanomain a subject has a specificity >70% at a classification threshold of 0.5CMCs/mL of blood. In additional embodiments, the method for diagnosingmetastatic melanoma in a subject has a specificity >80% at aclassification threshold of 0.5 CMCs/mL of blood. In additionalembodiments, the method for diagnosing metastatic melanoma in a subjecthas a specificity >90% at a classification threshold of 0.5 CMCs/mL ofblood.

From the foregoing description, it will be apparent that variations andmodifications can be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLES Example 1. Fluid Biopsy for Melanoma

This example describes a new high definition circulating tumor cell(HD-CTC) assay to identify CMCs in melanoma patients. Using thedescribed HD-CTC assay, CMCs can be characterized and identified by acombination of detectable markers and morphological characteristics. TheHD-CTC assay can quantify the small number of CMCs in the presence of amuch larger number of white blood cells.

HD-CTC technology is distinct in that it does not rely on anyprotein-based enrichment strategy to detect CTCs, but usesmulti-parametric computational analysis instead. All nucleated cells areretained and immunofluorescently labeled with tissue-specific monoclonalantibodies. Importantly, cells that do not meet specific selectioncriteria are not discarded. Therefore, each individual cell and itsassociated analysis data are catalogued in databases for subsequentre-analysis. Moreover, this strategy does not alter cell morphologyallowing detailed pathologic analysis of the CTC population that isknown to be very heterogeneous. This immunoenrichment-free strategy hasbeen proven to yield high sensitivity and specificity for CTC detectionin a variety of epithelial cancers, and results in substantially highersensitivity than the current standard and FDA-approved CellSearch®methodology.

The biomarker selected for the detection of CMCs in the present exampleis specific for chondroitin sulfate proteoglycan 4 (CSPG4), also knownas high molecular weight melanoma associated antigen (HMW-MAA) andmelanoma chondroitin sulfate proteoglycan (MCSP). CSPG4 is reported tobe highly expressed on melanoma cells in at least 80% of melanoma tumorsand has limited distribution in other tissues (Campoli et al., Crit RevImmunol. 2004; 24(4):267-96). CSPG4 is continuously expressed throughoutthe course of the disease, and does not appear to be affected by thetherapies used in melanoma (Yang et al., J Cell Biol. 2004 Jun. 21;165(6):881-91). It has also been used to identify metastatic melanomacells in sentinel lymph nodes by RT-PCR and immunohistochemistry (Gotoet al., Clin Cancer Res Off J Am Assoc Cancer Res. 2008 Jun. 1;14(11):3401-7). Additionally, soluble CSPG4 has been used to detect CMCsin scrum samples from melanoma patients (Vergilis et al., J InvestDermatol. 2005 September; 125(3):526-31), and immunomagnetic enrichmentof CMCs targeting CSPG4 has been shown (Ulmer et al., Clin Cancer ResOff J Am Assoc Cancer Res. 2004 Jan. 15; 10(2):531-7; Kitago et al.,Clin Chem. 2009 April; 55(4):757-64; Sakaizawa et al., Br J Cancer. 2012Feb. 28; 106(5):939-46). A panel of seven high-affinity monoclonalantibodies (mAbs) raised against distinct and distant epitopes of CSPG4are used. These mAbs provide reliable identification of different CMCssubpopulations based on CSPG4 protein expression, size and morphologyand show the phenotypic heterogeneity of CMCs within and across melanomapatients.

Methods Melanoma Cell Lines and Spiking Experiments

The three cell lines used in this example represent the major stages ofmelanoma progression: radial growth phase (WM1789), vertical growthphase (WM278) and metastasis (WM1617). All three melanoma cell lineswere purchased from the Wistar Institute Collection at the CorielInstitute for Medical Research, Camden, N.J., USA. The cells weremaintained in Tu2% melanoma growth medium, consisting of four parts ofMCDB153 (Sigma-Aldrich, Saint Louis, Mo. USA) and one part of L-15(invitrogen, Carlsbad, Calif., USA), supplemented with 1.68 mmol/Lcalcium chloride, 5 μg/ml insulin and 2% fetal bovine serum (Invitrogen,Carlsbad, Calif., USA). The culture medium was changed every 2 days. Todetermine expression of markers, melanoma cells were spiked intoperipheral blood mononuclear cells (PBMCs) in 1:100 ratio. To evaluatesensitivity, 0, 10, 50, 100 and 500 cells were spiked in three millionPBMCs. To mimic patient samples, the melanoma cells, removed from theculture plate with 0.02% Versene (Lonza, Walkersvill, Md., USA) topreserve membranous expression of CSPG4, were added to the PBMC pelletobtained after red blood cell lysis. The assay was repeated three timesto validate the reproducibility of the assay.

CSPG4 Monoclonal Antibodies

The CSPG4-specific mAb 149.53, 763.74, TP61.52, VF1-TP41.2, VF4-TP108,VF4-TP109.2, VT80.12 were developed and characterized as previouslydescribed (Campoli et al., Crit Rev Immunol. 2004; 24(4):267-96; Goto etal., Clin Cancer Res Off J Am Assoc Cancer Res. 2008 Jun. 1;14(11):3401-7, 21; Giacomini et al., J Immunol. 1985 July;135(1):696-702; and Temponi et al., Hybridoma 1989 February;8(1):85-95). The purity of mAb preparations was assessed by SDS-PAGEanalysis, and the activity of mAb preparations was monitored by testingwith CSPG4-bearing melanoma cells in a binding assay.

HD-CTC Assay for Melanoma

For establishing the CSPG4 staining protocol, preparations of the threemelanoma cell lines spiked with and without PBMCs from a normal donorwere used. To optimize the staining procedure, all relevant parametersof the protocol were evaluated as follows: staining before and afterslide freezing (see blood sample processing below); cell fixation was 20minutes at room temperature with 2% neutral buffered formalin solutionbefore or after freezing; total antibody concentration used was 0.07,0.15, 0.3, 0.6, 1.25, 2.5, 5, 7.5 and 12 μg/ml made in 10% goat serum;incubation times for primary antibodies were 40 min, 2 hours andovernight; incubation time for secondary antibody was 40 min. Theoptimal CSPG4 staining (low background, membranous staining in melanomacells and no staining in WBC) was determined to be as follows: After theslides were thawed and fixed with 2% neutral buffered formalin solution(100503, VWR, CA) non-specific binding sites were blocked with 10% goatserum (Millipore, Calif.). Slides were subsequently incubated withCSPG4-specific mAbs (5 μg/ml total concentration) and Alexa 647preconjugated anti-CD45 antibody (MCA87A647, AbD serotec, USA) for 40min at 37° C. After a washing step with PBS, slides were incubated withAlexa Fluor 555 goat antimouse IgG1 antibody (A21127, Life technologies,USA) for 20 min at 37° C. Cells were counterstained with Hoechst 33258(H1398, Life technologies, USA) for 30 min at room temperature andmounted with an aqueous mounting media. Slides of PBMCs with no addedtumor cells served as a negative control. Further characterization ofCSPG4 positive cells was performed using a mAb targeting HMB45 (clonegp-100, Dako, Denmark), a tissue-specific marker for melanocytes (AlexaFluor 488 goat antimouse IgG1 antibody (A21121, Life technologies,USA)).

Patient Population and Blood Sample Collection

All metastatic melanoma patients enrolled in this study were enrolledprospectively and signed consent forms for the use of blood samples andphysical and medical records. All samples were de-identified after theblood was drawn. 8 mi of peripheral blood was collected from 40metastatic melanoma patients in anti-coagulated Cyto-Chex® BCT tubes(Streck Innovations, Omaha, Nebr.) according to institution specificIRB-approved protocols. Samples from non-local sites (ComprehensiveCancer Centers of Nevada, Las Vegas, Nev.) were shipped overnight sothat the sample were received and processed within 24 h. Samples fromlocal sites (Pacific Oncology and Hematology, Encinitas, Calif.) wereheld at room temperature for 24 h to mimic samples coming from non-localsites. Blood specimens were also drawn from 10 normal blood donors(NBDs) from The Scripps Research Institute's Normal Blood Donor Service.The analysis of the samples was conducted with no previous knowledge ofpatient's disease status. Clinical data including age, date of initialdiagnosis, histology, LDH levels, performance status and BRAF statusamongst others were collected retrospectively.

Blood Sample Processing for HD-CMC Enumeration and Characterization

Whole blood specimens were prepared according the following method.Blood tubes were rocked for 5 min before a WBC count was performed usingthe Hemocue WBC system (HemoCue, Sweden). Based upon the WBC count, avolume of blood was subjected to an isotonic erythrocyte lysis withNH4Cl (ammonium chloride) buffer. After centrifugation, nucleated cellswere re-suspended in PBS to a final concentration of 4 million cells perml. plated as a monolayer on custom made glass slides and incubated for40 min at 37° C. The glass slides are the same size as standardmicroscopy slides, but the slides have a proprietary coating that allowsmaximal retention of live cells. Each slide can hold approximately threemillion nucleated cells; thus the number of slides obtained depended onthe patients' WBC count. After the incubation time, the slides arecoated with 7% BSA for 5 min and finally dried on a heat block at 37° C.for 12 min. The slides created for each patient are then stored at −80°C. for future experiments. For HD-CMC detection in melanoma patients,four slides holding approximately twelve million cells were used as atest.

Imaging and Technical Analysis

All nucleated cells in the specimen were imaged by a fluorescentautomated microscope. Each slide was scanned entirely at 10×magnification and produced 2304 images per channel used. The obtainedimages were analyzed using custom computer algorithms and the resultingHD-CMC cell candidates were identified based upon numerous measures,including CSPG4 and CD45 intensity, Hoechst positivity. and nuclear andcellular morphology. Finally, the HD-CMC candidates were evaluated bydirect review and classified as a HD-CMC or not based on immunophenotypeand cell morphology. Other cells related to CMCs but lacking essentialfeatures of a tumor cell were also tracked and classified. CMC countswere reported per milliliter of blood (CTCs ml⁻¹). The value iscalculated by counting the total number of nucleated cells on the glassslide used to isolate and detect CMCs and comparing it to the PBMC countin the patient's blood specimen. The ratio of counted nucleated cellsover the PBMCs per milliliter in the blood specimen determined thevolume of blood used per test (four slides). For this reason, fractionalvalues of CMCs ml⁻¹ are possible.

Re-Location and Re-Imaging

The HD-CMCs were relocated and reimaged using a macro written forImagePro Plus (Media Cybernetics, Bethesda, Md.). The images were takenat a fixed exposure intensity and gain at 40× magnification on a Nikon80i (Melville, N.Y.) epifluorescent microscope equipped with a QImagingRetiga EXi 12-bit monochrome CCD camera (QImaging, Surrey, BC, Canada).

Statistical Analysis

Relative CSPG4 values measured in cell lines were compared by one-wayANOVA with a Mann Whitney correction test using GraphPad Prism.

As CMC levels are not normally distributed, non-parametric tests wereused. Means between two groups were compared using the Mann-Whitneytest. One tailed P values of <0.05 were considered significant.

Results CSPG4 Protein Expression, Sensitivity and Specificity onMelanoma Cells

The expression of CSPG4 protein was first assessed by immunofluorescenceanalysis with the following CSPG4-specific mAbs 149.53, 763.74, TP61.5,VF1-TP41.2, VF4-TP108, VF4-TP109.2, and VT80.12 to verify expression,sensitivity and specificity of each specific mAb on three melanoma celllines under spiking conditions where melanoma cells were mixed withPBMCs from a normal donor control. CSPG4 protein expression was detectedon the surface of all three melanoma cell lines (FIG. 1A). Eachindividual mAb recognizes different and distant epitopes of CSPG4.Expression of the targeted epitopes recognized by mAb VT801.2 and TP61.6were more commonly expressed across cell lines, whereas those recognizedby mAb 149.53 and TP41.2 more heterogeneously distributed (FIG. 1B).This heterogeneous expression of CSPG4 epitopes provided the rationalefor the use of a combination of all 7 CSPG4-specific mAbs in this assay.The intensity of the staining provided by the combination of all 7CSPG4-specific mAbs was higher than those obtained using individual mAbsat the same concentration. PBMCs from a normal blood donor were used tonormalize signal intensities. Based on immunofluorescent parameters, anintact ID-CMC was defined as a cell that was: CSPG4 positive, CD45negative, with an intact non-apoptotic nucleus by Hoechst imaging.Positivity for CSPG4 was defined as the fluorescent signal being atleast 2 fold the background signal of surrounding PBMCs. Negativity ofCD45 was defined as having signal below visual detection under thecondition that 99% of all surrounding PBMCs were detectable globally.

HD-CMC Assay Linearity by Enumeration of Melanoma Cells Spiked in PBMCs

To test assay linearity using the combination of all 7 CSPG4-specificmAbs, serial dilutions of melanoma cells (0, 10, 50, 100 and 500) werespiked into approximately 3×10⁶ PBMC from a normal blood donor intriplicates and processed according to the ID-CMC assay. As displayed inFIG. 2, the number of WM1617, WM278 and WM1789 cells detected is plottedagainst expected cells. A percentage of detection of 97, 98.3 and 97.3with a linear detection coefficient of 0.99, 0.99 and 0.97 was obtainedusing WM1617 (FIG. 2A), WM278 (FIG. 2B) and WM1789 (FIG. 2C) cell lines,respectively.

Assay Specificity by Comparison of Normal Blood Donors and MelanomaPatients' Samples Using the HD-CMC Definition

To assess the specificity of the assay, 10 blood samples from normaldonors and 40 from melanoma patients were compared (FIG. 3).

Samples from normal donors were evaluated as a control populationconsisting of 6 females and 4 males with an age range of 40 to 79 years.A total of 105 candidate cells were found. Upon a post—classificationanalysis, 48% of cells (55 cells) did not meet one of the inclusioncriteria by having a CSPG4 signal below the cutoff and were easilyexcluded. Explicit review of the rest of cells (52% of total) revealed asimilar pattern, in that they were near to the lower limit for inclusionby one or more criteria. In general, these cells were CD45 negative, hadan intact nucleus, and had a CSPG4 signal intensity up to 7.8-foldbrighter than the surrounding PBMCs. However, this signal did not followthe CSPG4 pattern characterized by a signal distribution on the cellmembrane, and visually did not appear to be significantly brighter thansurrounding PBMCs by single channel fluorescent analysis. In addition tothat, analysis performed by a histopathologist confirmed that they weremorphologically different than surrounding PBMCs with different shapesand enlarged nuclei but they did not have a morphology compatible with amalignant phenotype based on by criteria used in standard diagnosticcytopathology such us enlarged size, architectural organization ofnucleus and cytoplasm, cytoplasmic shape, and nuclear shape.

Samples from 40 melanoma patients were also evaluated in parallel as atest population consisting in 15 females and 25 males with an age rangeof 45 to 91. In this patient group, 740 candidate cells were identified.Seventeen percent of them (124 cells) had a CSPG4 signal below thecutoff and were excluded. The remaining eighty three percent (616 cells)fulfill strictly all the inclusion criteria. On average, these cells hada mean CSPG4 intensity of 31.8, and a standard deviation of 49.3, 68%(415 cells) had a visually bright relative CSPG4 signal ranging from 8to 389 and 32% (198 cells) were near to the lower limit of CSPG4 signal,being just between 2- and 8-fold brighter than the surrounding PBMCs.Upon thorough post-classification review of these margin cells, amorphologically heterogeneous population of HD-CTCs was observed withinand across the patients. HD-CTCs had various cellular and nuclear shapesand sizes, and different CSPG4 patterns. Since cells in this range ofCSPG4 intensity were also detected in samples from normal blood donors,we assumed that normal blood cells may also exist among cells thatpopulate this group (CSPG4 low, CD45 negative, intact nucleus) inmelanoma patients. Excluding this group of cells by setting the CSPG4intensity cutoff at 8 would increase the specificity of the assay butwould decrease the sensitivity leading in false negative results. In theorder hand, including this group by keeping the cutoff at 2 would leadto false positive results. Image analyses of physical characteristics ofthese cells were made to determine quantitative differences that mayfunctionally contribute to improve inclusion criteria for a cell to bedefined as a HD-CMC.

Tailoring HD-CMC Definition

The HD-CTC platform allows for simultaneous cytomorphologic review offluorescent images for the individual channels with cell-by-cellautomatic annotation of data regarding size and fluorescent intensity ofobjects. Based on immunofluorescent parameters, an intact HD-CMC wasdefined as a cell that was: CSPG4 positive (at least 2-fold brighterthan surrounding PBMCs), CD45 negative, with an intact non-apoptoticnucleus by Hoechst imaging. In addition to these characteristics andbased on cytomorphologic parameters, HD-CMCs must be distinct fromnormal PBMCs, and must have a morphology that is compatible with amalignant phenotype by criteria used in standard diagnosticcytopathology such us enlarged size, architectural organization ofnucleus and cytoplasm, cytoplasmic shape, and nuclear shape. Upon reviewand quantification of physical parameters of all cells detected in bothnormal controls and melanoma patients. relative nuclear size was themost informative parameter among others. It was calculated as the ratioof individual nuclear sizes of candidate cell (in pixels) and the meannuclear size of surrounding PBMCs. Seventy percent of cells (516)detected in melanoma patients had a relative nuclear size smaller than2.5 with an average of 1.5. The remaining 30% (224 cells) had a relativenuclear size up to 13-fold larger than the surrounding PBMCs with anaverage close to 5. The results obtained from normal donor samplesshowed that 100% of cells had a nuclear size of at least 2.5-fold largerthan surrounding PBMCs, with an average of 5. Based on this evaluationwe defined a new exclusion criterion for those cells near to the lowerlimit of CSPG4 intensity. Thus, cells from patients that were CD45negative, CSPG4 low, had an intact nucleus and had a relative nuclearsize of 2.5 or larger were excluded. This population represented 54% ofcells (108) initially included in the low CSPG4 group based onimmunofluorescent parameters. After complete immunocytochemicaldetection and cytomorphical analysis of the HD-CMCs candidates, 84.9% ofthem were identified as HD-CMCs (CSPG4 mean 42.7±54.29).

Patient Demographics and HD-CMC Data

Between November 2010 and August 2013, 40 patients were recruited inthis study and their clinical data are shown in detail in Table 1. The40 patients comprised 25 males and 15 females, with a median age of 55.5years (range 45-91 years). All patients have metastatic disease, asdetermine by radiological and clinical criteria at the time of blooddraw. Metastatic sites included brain (12), lung (13), liver (8), bone(7), adrenal gland (4), subcutaneous (8) and skin (9). Thirty patientshad stage IV and ten patients had stage IIIC. BRAF mutational status wasassessed in 25 of patients and 11 of them had the mutation V600Edetected. Twenty-one patients with stage IV and one with stage IIIC haddied by the time of analysis, 3 were in complete remission, 13 werealive with disease, 2 were in progression and one was lost to follow-up.The follow-up period ranged from 0.08 to 22 months.

TABLE 1 Patient Characteristics and HD-CMC Data Survival Average afterCTC CMC/ml Patient collection LDH@ IUL Characteristics n(%) (range)number CMC/ml (days) draw N Status Stage Patients 40 15.0

Age (years) Median/range 55.5/45-91 Gender Female 15 (37.5)

Male 25 (62.5) 8.5 (0-13.4) Race Asian 1 (2.5)  16.5 White 35 (87.5)

Deceased IV Unknown 4 (10) 0.4 (0-1.1)

134.0 

141.4

Deceased IV BRAF status

ND ND Deceased IV Mutated 11 (27.5) 46.1 (0-358.1) 25

225 Deceased IV WT 14 (35) 1.2 (0-4.8)

ND ND Deceased IV ND/unknown 15 (37.5)

4.0

ND ND Deceased IV Number of

Alive stable IV CMC/ml disease =0 12 (30%)

Deceased IV 0.5-1 7 (17.5%) 0.7 (0.5-0.8)

Alive complete IIIC remission

Alive complete IV remission Type of primary

Alive stable IV melanoma disease cutaneous 30 (75) 20.0 (0-358.1)

Deceased IIIC Rectal 1 (2.5) 0  4 2.1 100 ND ND Deceased IV Unknown 5(12.5) 0.4 (0-1.1)

Deceased IV Primary Unknown 4 (10) 0.7 (0-2.1) 20 1.5

ND ND Alive stable IIIC disease Primary tumor 19 1.4 274 ND ND DeceasedIV histology Superficial 6 (15)

1.3

Deceased IV spreading Nodular 14 (37) 16.0 (0-134)

1.2

ND ND Alive stable IIIC disease Lentigo 1 (2.5) 0

1.2

Alive stable IV disease Spindle cell 3 (7.5) 0.4 (0-1.3) 10 1.1 431

225 Alive stable IV disease Unknown 16 (40) 23.1 (0-358.1) Site ofmetatasis Skin 9 (22.5) 7.1 (0-53.7) subcutaneous 8 (20) 0.8 (0-3.3)lung 13 (32.5)

liver 8 (20)

Bone 7 (17.5) 5.5 (0-3.3) Brain 12 (30) 11.9 (0-134) Adrenal 4 (10) 0.8(0-2.4) Stage IIIC 10 (25) 0.8 (0-3.1) IV 30 (75) 20.1 (0-358.1)Survival status Deceased 22 (55) 26.6 (0-358.1) Alive 17 (42.5) 0.9(0-3.6) stable 13 (76.4) 0.9 (0-3.6) complete 3 (17.6) 2.3 (0.8-3.1)remission Progression 2 (11.7)

Lost to follow 1 (2.5) up

indicates data missing or illegible when filed

Prevalence of HD-CMC in Metastatic Melanoma Patients

Following the strict inclusion criteria, we detected 1 and 241 HD-CMC in28 (70%) of 40 metastatic melanoma patients. The number of HD-CMCsranged between 0.5 and 358.1/mL (mean 15.1), while no HD-CMCs weredetected in the blood of normal blood donors (FIG. 4A). Twenty one ofthe positive patients (75%) had ≥1 HD-CTCs ml-1; 14 (50%)≥2 HD-CTCsml-1, 4 (14.3%)≥10 HD-CTCs mi-1 and 2 (7%)≥100 HD-CTCs ml-1. The CSPG4signal intensity within the HD-CMC population varied within and acrosspatients (FIG. 4B). Five patients (2, 3, 9, 31 and 38) accounted onlywith CSPG4 bright HD-CMCs, 14 patients (4, 8, 11, 13-15, 17-18, 21,24-25, 28, 35 and 39) had only CSPG4 dim HD-CMCs, and 9 patients (5, 6,10, 19, 20, 30, 33, 37 and 40) had both CSPG4 bright and CSPG4 lowHD-CMCs. In the latter group, 4 patients (5, 30, 33 and 37) had mostlyGSPG4 bright cells with a percentage ranging from 67 to 95%, 2 patients(6 and 10) had equal numbers and 3 patients (19, 20 and 40) had mostlyCSPG4 low HD-CMCs ranging from 66% to 87.5%. FIG. 4D shows thecytomorphology and immunophenotype of 2 representative HD-CMCs from thethree melanoma patients (patient 5, 30 and 37). To evaluatecytomorphologic heterogeneity on HD-CMCs, we analyzed the cells found inthe two patients (30 and 37) with more than 100 HD-CTCs ml-1 to delivermeaningful results (FIGS. 4C, 4D and 4E). HD-CMC shapes varied withinand across patients. Specially, most of the cells from patient 30 werehigh pleomorphic in shape and presented polygonal nuclei (FIG. 4C).Roundness mean was 1.01 in average for patient's PBMC, and 1.1 and 1.04for HD-CMCs in patient 30 and 37 respectively, indicating that HD-CMCswere slightly oblong in shape compared to the surrounding PBMCs (FIG.4D). Area plots indicate that HD-CMCs in melanoma patient 30 and 37 weresignificantly larger than their corresponding PBMCs (Mean area: 2×10⁻³vs. 1×10⁻³, P<0.0001 and 1.7×10⁻³ vs. 1×10⁻³, P<0.0001, respectively)(FIG. 4D). In most of patients, only single cells were detected, exceptfor 4 patients containing from two-cell to four-cell clusters.

Characterization of HD-CMCs with HMB-45

Expression of HMB45 in combination with CSPG4 was first evaluated onmelanoma cell lines and was detected in the cytoplasm of WM1789 andWM1617 cells lines, while no detection was observed in WM278 (data notshown). Same analysis was performed in the cohort of 40 metastaticmelanoma patients (1 slide each). Cells were first selected based on thepreviously established inclusion criteria for an object to be selectedas a HD-CMC. All HD-CMCs detected were then exhaustively analyzed andclassified as either HMB45 positive (HMB45+) or negative (HMB45−). FIG.5A shows the distribution of 124 HD-CMCs found in 40 melanoma patientsthat were either CSPG4⁺/HMB-45⁻ (left) or CSPG4⁺/HMB-45⁺ (right).Sixty-one of 124 cells (49%) were CSPG4^(∘)/HMB-45⁻ while the remaining68 cells (51%) were CSPG4⁺/HMB-45⁺. To evaluate signal heterogeneity ofHD-CMCs within and across patients, we analyzed cells found in the twomelanoma patients (30 and 37) who accounted with more than 100 HD-CTCsmi-1 each to obtain significant results. In patient number 30, 58 cellswere reviewed. In general, HD-CMCs from this patient had a high relativeCSPG4 intensity (mean 62.9). Only 33 of them (57%) were positive forboth CSPG4 and HMB-45. Mean relative HMB-45 intensity of double positivecells was 20.7. In patient number 37, 37 cells were evaluated. In thiscase, twenty-four cells (68%) were positive for both CSPG4 and HMB-45.Mean relative CSPG4 intensity was 16.3, while mean relative HMB-45intensity was 34.6 in the positive setting. FIG. 5B shows theimmunologic heterogeneity observed between HD-CMCs within and acrossthese two patients. Despite the low sensitivity of the HMB-45 markerobserved in our assay, its high specificity supported the inclusion ofthose HD-CMCs accounting with low CSPG4 signal intensity, especially inpatient number 37 where 26% of HD-CMCs had a relative CSPG4 intensitysignal close to the lower limit cutoff.

CTC Levels and Clinical Outcome of Melanoma Patients

One aspect of this study was to adapt HD-CTC technology for thedetection of CMCs. The number of patients in this study (n=40) was thusnot powered for survival analysis nor was the sampling of bloodcontrolled for line of therapy. Nevertheless, there was an associationbetween the number of CMC per ml of blood and the short survivalobserved in these patients. A receiver operating characteristic (ROC)curve was constructed using the results from melanoma patients (n=39). Avalue of 10.7 HD-CTCs ml-1 was determined from this cohort data. OS timefor those patients with <10.7 ID-CTCs ml-1 was 315.9 days and wassignificantly longer than the median OS time for those patients with≥10.7 CTCs ml-1, 18 days (FIG. 6).

In summary, the data shown herein, for the first time, demonstrates thatCMCs are identified by the HD-CTC technology in 70% of metastaticmelanoma patients and that CMCs constitute a very heterogeneouspopulation in terms of CSPG4 expression and cell morphology within andacross melanoma patients. The enrichment-free methods described enabledirect immunocytochemical detection of CMCs using a panel of 7 CSPG4mAbs that recognizes distinct and distant epitopes of CSPG4.Additionally, for the first time. expression of CSPG4 protein has beenshown in WM1617. WM278 and WM1789 melanoma cell lines. Because CSPG4epitope expression is unknown in CMCs and known to be heterogeneous inmelanoma cell lines. use of a panel of CSPG4-specific mAbs for CMCsdetection in melanoma patients can be an efficient strategy to detectCMCs. Additionally, use of a combination of CSPG4-specific mAbs markedlyincreased the intensity of the staining of melanoma cell lines. We alsofound that immunocytochemical characterization with HMB-45 could aid inthe identification of HD-CMCs with low CSPG4 signal. As for themorphology of CMCs, in general, most had an enlarged size and weremorphologically different than surrounding PBMCs. CMCs were alsomorphologically different within and across patients going fromepitheliod cells with oval nuclei to polygonal cells with polarizednuclei. However, despite these cells displaying a wide range of CSPG4intensities, the majority of the cells had a similar relative nuclearsize, averaging 1.3 times larger than surrounding PBMCs. Interestingly,a subset of cells with low CSPG4 intensity had a range of nuclear sizesand were, on average, five-times larger than PBMCs. Similar large cellswith low CSPG4 signal were also found in some NBD. Nevertheless, we wereable to define strict inclusion criteria to safely exclude thisborderline cells that were eventually recognized by a histopathologistas enlarged hematopoietic cells. Lastly, we observed that patients withmore than 10.6 CMCs per ml of blood expired within 30 days of bloodsample collection.

Example 2. DNA Copy Number Variation Analysis of CMCs

This example describes the DNA analysis of single CMCs that wereisolated from two melanoma patients identified in Example 1.

CMC extraction and copy number variation (CNV) analysis were conductedby relocating CMCs on the glass slide and reimaging at 40× for detailedcytomorphometric analysis as previously described in Marrinucci D. etal., 2012, Phys. Biol. 9 016003. CMCs were then captured bymicromanipulation and whole genome amplification and CNV analysis wereperformed as previously described in Dago E. et al., 2014, PLoS ONE 9e10177.

DNA CNVs were assessed in single CMCs, WBCs and ‘excluded candidatecells’ isolated from melanoma patient #30 (40 cells) and #37 (23 cells)(FIGS. 7A-7F). Chromosomal alterations were found in 100% of the CMCsanalyzed. A unique clonal population (38 CMCs) in patient #30 and twoclonal populations (18 CMCs) in patient #37 were observed. Chromosomalgains and deletions of chr5, 7, 9, 10, 12, 17, and 19 were detected inboth patients.

Candidate genes encoding components of commonly altered pathways inmelanoma were located at these amplified/deleted areas. For example, theamplification of mixed-lineage leukemia 3 (MLL3) in chromosome 7, animportant histone regulator gene, and the loss of cyclin-dependentkinase inhibitor 2A (CDKN2A), a tumor suppressor gene that regulates thepRB and p53 pathways (Flores J F el al., 1996, Cancer Res. 56: 5023-32;and Hodis E et al., 2012, Cell 150: 251-63), were found to be present inboth patients, along with an increase of a segment on chromosome 5pcontaining telomerase reverse transcriptase (TERT) locus, which encodesthe catalytic protein subunit of the telomerase (Hodis E et al., supra).The loss of phosphatase and tensin homolog (PTEN), responsible for thenegative regulation of the PI3K/AKT pathway (Paraiso K H T et al., 2011,Cancer Res. 71: 2750-60) was found only in patient #30. In patient #37,two CMC populations (clone A and B) were identified (FIG. 7B). Mousedouble minute 2 homolog (MDM2), an important negative regulator of thep53 tumor suppressor (Muthusamy V et al., 2006, Genes. ChromosomesCancer 45: 447-54), was amplified in all CMCs from both clones and havemore than 20 copies each. Amplification of BRAF (Shi H et al., 2012,Nat. Commun. 3: 724), which regulates the MAPK signaling pathway, wasidentified in all CMCs from clone A. Kirsten rat sarcoma viral oncogenehomolog (KRAS), involved primarily in regulating cell division, was onlyamplified in clone B. No chromosomal alterations were detected in theWBCs or ‘excluded candidate cells’ (data not shown). Thus, the aboveanalysis of single CMCs identified deletions of CDKN2A and PTEN, andamplification(s) of TERT, MLL3, BRAF, KRAS and MDM2.

The above results also showed a heterogeneous BRAF status between CMCsand matched tumor tissues as well as within the CMC population inindividual patients. This suggests that the complex genomic diversity ofmelanoma is also illustrated in the CMC population.

Limited heterogeneity of CMCs in terms of chromosomal CNVs was alsofound when hierarchical cluster analysis was performed, consistent withprevious reports. However, mutational analysis was not performed and itis possible that CMCs carry private mutations.

One important result associated with the genomic profiling shown aboveis the identification of novel altered chromosomal regions in CMCs inaddition to markers of clinical significance known in melanoma. Acomplete deletion of CDKN2A together with the amplification of MDM2 inpatient #37 suggest that the p53 pathway is inactivated (Zhang Y et al.,1998, Cell 92: 725-34) in this CMC population. Consistent with this,mutation analysis of cutaneous melanoma sequencing data showed that MDM2amplification (4%) and CDKN2A deletion or mutation (42%) occur in around55% of melanoma cases. Recent studies have supported the ability torestore the apoptotic function of p53 as a parallel therapeutic strategyalongside BRAFV600E inhibition in the treatment of melanoma (Lu M etal., 2013, Cancer Cell 23: 618-33; and Lu M et al., 2014, FEBS Lett.558: 2616-21). Moreover, PTEN deletion in all CMCs from patient #30 andBRAF amplification in the CMCs populating clone A in patient #37 havebeen described as two distinct mechanisms of drug resistance after BRAFinhibitor therapy (Nazarian R et al., 2010, Nature: 468 973-7) (Shi H etal., 2012, Nat. Commun. 3: 724) and could, in part, explain why thosepatients progressed. Importantly, eight novel chromosomal amplificationsare shown in chr12, 17 and 19 including cancer genes such as AKT2,PIK3C2 and BRIP1.

In light of the above results, screening for targetable genomicalternations at the single cell level can identify subpopulations ofpatients who will benefit from molecularly targeted therapies and allowtheir monitoring in real time.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1. A method for identifying circulating melanoma cells (CMCs) in abiological sample comprising: (a) contacting a biological sample ofnon-enriched blood with one or more detectable agents, wherein at leastone of said one or more detectable agents is specific for a biomarker ofCMCs; (b) determining the presence or absence of said one or moredetectable agents in or on nucleated cells in the sample; and (c)assessing the morphology of the nucleated cells having said one or moredetectable agents, wherein the CMCs are identified based on acombination of the presence or absence of said one or more detectableagents and morphological characteristics of the nucleated cells.
 2. Themethod of claim 1, wherein said one or more detectable agents comprise aimmunofluorescent marker.
 3. The method of claim 2, wherein saidimmunofluorescent maker is an antibody or functional fragment thereofthat specifically binds to chondroitin sulfate proteoglycan 4 (CSPG4) orpremelanosome protein (Pmel17), or S100 calcium-binding protein A1(S100A1).
 4. (canceled)
 5. The method of claim 1, wherein said one ormore detectable agents comprise two, three, four, five, six, seven ormore immunofluorescent markers.
 6. The method of claim 1, wherein saidone or more detectable agents comprise a nucleic acid specific stain. 7.(canceled)
 8. The method of claim 1, wherein said one or more detectableagents comprise an immunofluorescent marker for white blood cells (WBCs)9. The method of claim 8, wherein said immunofluorescent marker for WBCsis an antibody specific for cluster of differentiation 45 (CD45). 10.The method of claim 1, wherein step (b) and/or (c) are performed byautomated fluorescent microscopy.
 11. The method of claim 1, whereinsaid determining the presence or absence of said one or more detectableagents comprises comparing distinct immunofluorescent staining of CMCswith distinct immunofluorescent staining of white blood cells (WBCs).12. The method of claim 11, wherein said immunofluorescent staining ofCMCs is positive for an antibody or functional fragment thereof thatspecifically binds to CSPG4 and is detectable at a standard deviation ofthe mean (SDOM) of greater than or equal to
 2. 13. The method of claim11, wherein said immunofluorescent staining of CMCs is negative for anantibody or functional fragment thereof that specifically binds to CD45.14. (canceled)
 15. The method of claim 1, wherein said morphologicalassessment comprises comparing the morphological characteristics of CMCswith the morphological characteristics of surrounding white blood cells(WBCs).
 16. The method of claim 15, wherein said morphologicalcharacteristics comprise nucleus size, nucleus shape, cell size, cellshape or nuclear to cytoplasmic ratio.
 17. The method of claim 16,wherein a nuclear to cytoplasmic ratio of less than 2.5 indicates thepresence of a CMC.
 18. The method of claim 1, further comprisingobtaining a white blood cell (WBC) count for the sample.
 19. The methodof claim 1, further comprising lysing erythrocytes in the sample. 20.The method of claim 1, further comprising depositing nucleated cellsfrom the sample as a monolayer on a glass slide.
 21. The method of claim20, comprising depositing about 3 million cells from the sample ontosaid glass slide.
 22. A method for diagnosing metastatic melanomacomprising: (a) contacting a biological sample of non-enriched bloodwith one or more detectable agents, wherein said sample was obtainedfrom a subject suspected of having metastatic melanoma or diagnosed withhaving melanoma, wherein at least one of said one or more detectableagents is specific for a biomarker of circulating melanoma cells (CMCs);(b) determining the presence or absence of said one or more detectableagents in or on nucleated cells present in the sample; (c) assessing themorphology of the nucleated cells having said one or more detectableagents; and (d) identifying the presence of CMCs in the sample based ona combination of the presence or absence of said one or more detectableagents and morphological characteristics of the nucleated cells, whereinthe subject is diagnosed with metastatic melanoma when a predeterminednumber of CMCs is present in the sample. 23-42. (canceled)
 43. Themethod of claim 22, wherein said predetermine number of CMCs is at least0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10, 20, 50, 100, 200, 300, 400or 500 CMCs per ml of sample.